Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches.

Anticytotoxic T lymphocyte-associated protein 4 (CTLA4) antibodies have shown potent antitumor activity, but systemic immune activation leads to severe immune-related adverse events, limiting clinical usage. We developed novel, conditionally active biologic (CAB) anti-CTLA4 antibodies that are active only in the acidic tumor microenvironment. In healthy tissue, this binding is reversibly inhibited by a novel mechanism using physiological chemicals as protein-associated chemical switches (PaCS). No enzymes or potentially immunogenic covalent modifications to the antibody are required for activation in the tumor. The novel anti-CTLA4 antibodies show similar efficacy in animal models compared to an analog of a marketed anti-CTLA4 biologic, but have markedly reduced toxicity in nonhuman primates (in combination with an anti-PD1 checkpoint inhibitor), indicating a widened therapeutic index (TI). The PaCS encompass mechanisms that are applicable to a wide array of antibody formats (e.g., ADC, bispecifics) and antigens. Examples shown here include antibodies to EpCAM, Her2, Nectin4, CD73, and CD3. Existing antibodies can be engineered readily to be made sensitive to PaCS, and the inhibitory activity can be optimized for each antigen's varying expression level and tissue distribution. PaCS can modulate diverse physiological molecular interactions and are applicable to various pathologic conditions, enabling differential CAB antibody activities in normal versus disease microenvironments.

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies.

A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human regions was used to isolate low-affinity antibodies to human ß nerve growth factor (hßNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K(D). This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.

A novel conditional active biologic anti-EpCAM x anti-CD3 bispecific antibody with synergistic tumor selectivity for cancer immunotherapy.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that plays several roles in cancer biology. EpCAM is an attractive therapeutic target because of its expression in most solid tumors. However, targeting EpCAM has been challenging because it is also highly expressed in normal epithelial tissues. Initial attempts to develop EpCAM-specific T-cell engagers were unsuccessful due to severe cytokine release effects, as well as serious on-target, off-tumor drug-related toxicities. We developed novel, conditionally active biological (CAB) bispecific antibodies that bind to both EpCAM and CD3 in an acidic tumor microenvironment. In healthy tissues, binding to EpCAM and CD3 is greatly reduced by a novel, dual CAB selection, where each binding domain is independently blocked by the presence of physiological chemicals known as Protein-associated Chemical Switches (PaCS). The CAB anti-EpCAM T-cell engagers displayed the anticipated bispecific binding properties and mediated the potent lysis of EpCAM-positive cancer cell lines through the recruitment of T cells in the tumor microenvironment. Xenograft studies showed that the efficacy of CAB bispecific antibodies is similar to that of a non-CAB anti-EpCAM bispecific antibody, but they have markedly reduced toxicity in non-human primates, indicating an unprecedentedly widened therapeutic index of over 100-fold. These preclinical results indicate that the dual CAB bispecific antibody is potentially both a powerful and safe therapeutic platform and a promising T cell-engaging treatment for patients with EpCAM-expressing tumors.

Development of a novel conditionally active EpCAM-specific T-cell engager with enhanced safety and tolerability for treatment of solid tumors.

The Use of Somatic Hypermutation for the Affinity Maturation of Therapeutic Antibodies.

The engineering of antibodies and antibody fragments for affinity maturation, stability, and other biophysical characteristics is a common aspect of therapeutic development. Maturation of antibodies in B cells during the adaptive immune response is the result of a process called somatic hypermutation (SHM), in which the activation-induced cytidine deaminase (AID) acts to introduce mutations into immunoglobulin (Ig) genes. Iterative selection and clonal expansion of B cells containing affinity-enhancing mutations drive an increase in the overall affinity of antibodies. Here we describe the use of SHM coupled with mammalian cell surface display for the maturation of antibodies in vitro and the complementarity of these methods with the mining of immune lineages using next-generation sequencing (NGS).

Role of RANK ligand in mediating increased bone resorption in early postmenopausal women.

Studies in rodents have implicated various cytokines as paracrine mediators of increased osteoclastogenesis during estrogen deficiency, but increases in RANKL, the final effector of osteoclastogenesis, have not been demonstrated. Thus, we isolated bone marrow mononuclear cells expressing RANKL on their surfaces by two-color flow cytometry using FITC-conjugated osteoprotegerin-Fc (OPG-Fc-FITC) as a probe. The cells were characterized as preosteoblastic marrow stromal cells (MSCs), T lymphocytes, or B lymphocytes by using Ab's against bone alkaline phosphatase (BAP), CD3, and CD20, respectively, in 12 premenopausal women (Group A), 12 early postmenopausal women (Group B), and 12 age-matched, estrogen-treated postmenopausal women (Group C). Fluorescence intensity of OPG-Fc-FITC, an index of the surface concentration of RANKL per cell, was increased in Group B over Groups A and C by two- to threefold for MSCs, T cells, B cells, and total RANKL-expressing cells. Moreover, in the merged groups, RANKL expression per cell correlated directly with the bone resorption markers, serum C-terminal telopeptide of type I collagen and urine N-telopeptide of type I collagen, in all three cell types and inversely with serum 17beta-estradiol for total RANKL-expressing cells. The data suggest that upregulation of RANKL on bone marrow cells is an important determinant of increased bone resorption induced by estrogen deficiency.

Regulation of protein catabolism by muscle-specific and cytokine-inducible ubiquitin ligase E3alpha-II during cancer cachexia.

The progressive depletion of skeletal muscle is a hallmark of many types of advanced cancer and frequently is associated with debility, morbidity, and mortality. Muscle wasting is primarily mediated by the activation of the ubiquitin-proteasome system, which is responsible for degrading the bulk of intracellular proteins. E3 ubiquitin ligases control polyubiquitination, a rate-limiting step in the ubiquitin-proteasome system, but their direct involvement in muscle protein catabolism in cancer remains obscure. Here, we report the full-length cloning of E3alpha-II, a novel "N-end rule" ubiquitin ligase, and its functional involvement in cancer cachexia. E3alpha-II is highly enriched in skeletal muscle, and its expression is regulated by proinflammatory cytokines. In two different animal models of cancer cachexia, E3alpha-II was significantly induced at the onset and during the progression of muscle wasting. The E3alpha-II activation in skeletal muscle was accompanied by a sharp increase in protein ubiquitination, which could be blocked by arginine methylester, an E3alpha-selective inhibitor. Treatment of myotubes with tumor necrosis factor alpha or interleukin 6 elicited marked increases in E3alpha-II but not E3alpha-I expression and ubiquitin conjugation activity in parallel. E3alpha-II transfection markedly accelerated ubiquitin conjugation to endogenous cellular proteins in muscle cultures. These findings show that E3alpha-II plays an important role in muscle protein catabolism during cancer cachexia and suggest that E3alpha-II is a potential therapeutic target for muscle wasting.

Bench to bedside: elucidation of the OPG-RANK-RANKL pathway and the development of denosumab.

Bone is a complex tissue that provides mechanical support for muscles and joints, protection for vital organs, a mineral reservoir that is essential for calcium homeostasis, and the environment and niches required for haematopoiesis. The regulation of bone mass in mammals is governed by a complex interplay between bone-forming cells termed osteoblasts and bone-resorbing cells termed osteoclasts, and is guided physiologically by a diverse set of hormones, cytokines and growth factors. The balance between these processes changes over time, causing an elevated risk of fractures with age. Osteoclasts may also be activated in the cancer setting, leading to bone pain, fracture, spinal cord compression and other significant morbidities. This Review chronicles the events that led to an increased understanding of bone resorption, the elucidation of the signalling pathway mediated by osteoprotegerin, receptor activator of NF-κB (RANK) and RANK ligand (RANKL) and its role in osteoclast biology, as well as the evolution of recombinant RANKL antagonists, which culminated in the development of the therapeutic RANKL-targeted antibody denosumab.

Surrogate approaches in development of monoclonal antibodies.

When cross-reactivity of a lead antibody across species is limited, antibody development programs require the generation of surrogate molecules or surrogate animal models necessary for the conduct of preclinical pharmacology and safety studies. When surrogate approaches are employed, the complexities and challenges for translation of preclinical safety and efficacy results to the clinic are undoubtedly enhanced. Because there are no currently established criteria or regulatory guidance regarding the application of surrogate approaches, a science-based strategy for translation of preclinical information to the clinic is vital for effective development of the lead antibody.

Denosumab, a fully human monoclonal antibody to RANKL, inhibits bone resorption and increases BMD in knock-in mice that express chimeric (murine/human) RANKL.

RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG-Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG-Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock-in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting "huRANKL" mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG-Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.

Osteoclast differentiation and activation.

Osteoclasts are specialized cells derived from the monocyte/macrophage haematopoietic lineage that develop and adhere to bone matrix, then secrete acid and lytic enzymes that degrade it in a specialized, extracellular compartment. Discovery of the RANK signalling pathway in the osteoclast has provided insight into the mechanisms of osteoclastogenesis and activation of bone resorption, and how hormonal signals impact bone structure and mass. Further study of this pathway is providing the molecular basis for developing therapeutics to treat osteoporosis and other diseases of bone loss.

RANK-L and RANK: T cells, bone loss, and mammalian evolution.

TNF and TNFR family proteins play important roles in the control of cell death, proliferation, autoimmunity, the function of immune cells, or the organogenesis of lymphoid organs. Recently, novel members of this large family have been identified that have critical functions in immunity and that couple lymphoid cells with other organ systems such as bone morphogenesis and mammary gland formation in pregnancy. The TNF-family molecule RANK-L (RANK-L, TRANCE, ODF) and its receptor RANK are key regulators of bone remodeling, and they are essential for the development and activation of osteoclasts. Intriguingly, RANK-L/RANK interactions also regulate T cell/dendritic cell communications, dendritic cell survival, and lymph node formation; T cell-derived RANK-L can mediate bone loss in arthritis and periodontal disease. Moreover, RANK-L and RANK are expressed in mammary gland epithelial cells, and they control the development of a lactating mammary gland during pregnancy and the propagation of mammalian species. Modulation of these systems provides us with a unique opportunity to design novel therapeutics to inhibit bone loss in arthritis, periodontal disease, and osteoporosis.